Direct Calorimetric Studies on the Heats of Ionization of Oxygenated and Deoxygenated Hemoglobin

Abstract

Abstract The total heats of ionization, Qo and Qr, of bovine, human, and horse oxygenated and deoxygenated hemoglobin (O2Hb and Hb) have been directly measured by the rapid calorimetric method over the pH range from 5.7 to 9.0, at 12–28°. The most extensive determinations have been those on bovine hemoglobin: above about pH 6.6 the thermal titration curve for Hb lies systematically above that for O2Hb by about 600 cal, this difference presisting practically unchanged up to the most alkaline pH (8.7) studied. The two thermal titration curves cross at approximately pH 6.3, below which the O2Hb curve lies above the Hb curve by an increasing amount (up to 1,000 cal). The fact that Qr remains greater than Qo at pH 8.7, at which the absolute value of Qr is about 11,000 cal, implies that the heme-linked group, which ionizes in this pH range in the case of Hb, must have a heat of ionization, Qr, of around 11,000 cal. This figure, which was confirmed by an approximate method of calculation, lies outside the range usually attributed to the heat of ionization of imidazole or its derivatives. There is some indication, from a comparison of the difference between the two thermal titration curves for human Hb and O2Hb at approximately pH 7.3, that (Qr - Qo) is of the order of 4,000 cal, Qo being the heat of ionization of the corresponding heme-linked group in O2Hb. The results thus support the conclusions reached in the adjoining paper by Rossi-Bernardi and Roughton on the effect of temperature on the oxygen-linked ionizations of hemoglobin. The relation of the present studies to the cognate effects of pH on the heat of oxygenation of hemoglobin is briefly indicated.